1. Field of the Invention
The present invention relates to the application of acridinium compounds with certain specific structural features in homogeneous assays. Important structural features that are necessary to ensure light emission at mild pH are disclosed herein.
2. Background of the Invention
Chemiluminescent acridinium esters (AE) are extremely useful labels that have been used extensively in immunoassays and nucleic acid assays. U.S. Pat. Nos. 4,745,181; 4,918,192; 5,110,932; 5,241,070; 5,538,901; 5,663,074 and 5,656,426 disclose a variety of stable acridinium esters with different functional groups for conjugation to a variety of biologically active molecules referred to as analytes.
U.S. Pat. No. 5,656,426 discloses a hydrophilic acridinium ester with increased quantum yield. Considerable effort has also been directed towards the design of acridinium esters whose emission wavelength can be altered by either incorporating unique structural features in the acridinium ester or by employing the principle of energy transfer. See U.S. Pat. Nos. 5,395,752; 5,702,887; 5,879,894; 6,165,800; and 6,355,803.
Acridinium sulfonamides are another class of chemiluminescent compounds where the substituted phenolic leaving group is replaced with a substituted sulfonamide. The synthesis and applications of these acridinium compounds in heterogeneous assays have been described in the prior art: Adamczyk et al, Tetrahedron, vol. 55, pages 10899-10914 (1999); Mattingly, J. Biolumin. Chemilumin., vol. 6, pages 107-114 (1991); and Adamcyzk et al, Bioconjugate Chem. vol. 11, pages 714-724 (2000).
Mechanistically, acid treatment converts the pseudo-base form of the acridinium compound to the acridinium ester which can then participate in the chemiluminescent reaction with hydrogen peroxide. The addition of alkali serves not only to neutralize the acid but also to raise the pH of the reaction medium for the ionization of hydrogen peroxide.
The relatively harsh reagents with strong acidic pHs on the order of less than 2 and strong basic pHs on the order of greater than 12 that are required for triggering to chemiluminescence as described above are detrimental to the preservation of binding complexes such as antibody-hapten complexes or nucleic acid hybrids. This is not a problem in a heterogeneous assay format where signal generation is typically performed at the end of the assay and unbound tracer and/or interfering substances have been removed.
A homogeneous assay is an analytical method where measurement of a substance of interest is performed without any separation procedures. In a homogeneous assay format, in order to detect the occurrence of binding events using chemiluminescent acridinium compounds as tags, light generation must occur under milder pH conditions because harsh pH conditions are detrimental to the preservation of binding complexes. Additionally, a mechanism to distinguish between bound and unbound tracer or analyte is needed because no separation is performed in a homogeneous assay. These constraints have hampered the utility of acridinium compounds in homogeneous assays.
The only homogeneous assays using acridinium compounds that are believed to have been described in the literature are “hybridization protection assays”. See Nelson et al, Biochemistry vol. 35, pages 8429-8438 (1996). In these assays, the acridinium ester portion of an acridinium ester-labeled nucleic acid probe is selectively decomposed by hydrolysis when it is not hybridized to a target. Hybridization of the probe to its target results in protection of the acridinium ester from hydrolysis thereby enabling the distinction between hybridized versus non-hybridized DNA. By employing acridinium esters with similar hydrolysis rates but different time-dependent light emission profiles, homogeneous multi-analyte assays were devised to detect nucleic acids.
In Nelson et al., the structure of the acridinium ester is not being altered to enable light emission under mild conditions. Rather, the Nelson et al. assay takes advantage of the different rates of hydrolysis (degradation) of the acridinium ester when a nucleic acid labeled with the acridinium ester is either free or is hybridized to the complementary sequence of the acridinium ester-labeled nucleic acid. Thus, the property of the nucleic acid, whether or not it is hybridized to its complementary sequence, is being used to alter the rate of degradation of the acridinium ester.
Fluorescence Resonance Energy Transfer (FRET) is a well-known phenomenon that has been widely used to study proximity effects in biomolecules. In FRET, an electronically excited fluorescent donor molecule transfers its electronic energy to a second, acceptor molecule through dipole-dipole coupling. This energy transfer causes fluorescence quenching of the donor. If the acceptor is fluorescent, its fluorescence is then observed.
The efficiency of energy transfer is inversely proportional to the sixth power of the distance separating the donor and acceptor fluors and also depends directly on the fluorescent quantum yield of the donor and the extinction coefficient of the acceptor at the wavelength of maximal emission of the donor. Because of the distance dependence, FRET is normally not observed at distances >10 nm. Homogeneous immunoassays based on chemiluminescence energy transfer have also been described using isoluminol as the chemiluminescent donor and fluorescein as the acceptor. See Patel et al, Clin. Chem. vol. 29/9, pages 1604-1608 (1983). Assays for small analytes such as progesterone as well as protein antigens were constructed using the isoluminol-fluorescein, donor-acceptor pair presumably because chemiluminescence from isoluminol can be triggered under mild conditions.